Abstract
Purified human liver carboxylesterase (hCE-1) catalyzes the hydrolysis of cocaine to form benzoylecgonine, the deacetylation of heroin to form 6-acetylmorphine, and the ethanol-dependent transesterification of cocaine to form cocaethylene. In this study, the binding affinities of cocaine, cocaine metabolites and analogs, heroin, morphine, and 6-acetylmorphine for hCE-1 were evaluated by measuring their kinetic inhibition constants with 4-methylumbelliferyl acetate in a rapid spectrophotometric assay. The naturally occurring (R)-(−)-cocaine isomer displayed the highest affinity of all cocaine and heroin analogs or metabolites. The pseudo- or allopseudococaine isomers of cocaine exhibited lower affinity indicating that binding to the enzyme is stereoselective. The methyl ester, benzoyl, and N-methyl groups of cocaine play important roles in binding because removal of these groups increasedKi values substantially. Compounds containing a variety of hydrophobic substitutions at the benzoyl group of cocaine bound to the enzyme with high affinity. The highKi value obtained for cocaethylene relative to cocaine is consistent with weaker binding to the esterase and a longer elimination half-life reported for the metabolite. The spectrophotometric competitive inhibition assay used here represents an effective method to identify drug or environmental esters metabolized by carboxylesterases like hCE-1.
Footnotes
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Send reprint requests to: Dr. William F. Bosron, Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, 635 Barnhill Drive, MS 405, Indianapolis, IN 46202-5122, wbosron{at}iupui.edu.
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This work was supported by RO1 DA06836 and fellowships to M.R.B. from T32 AA07462.
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↵2 (R)-(+)-pseudococaine is the C2 epimer of (R)-(−)-cocaine, which is found in the leaves ofErythroxylon coca.
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↵3 (S)-(+)-cocaine is the unnatural enantiomer of (R)-(−)-cocaine, and (S)-(−)-pseudococaine is the C2 epimer of (S)-(+)-cocaine.
- Abbreviations used are::
- KM
- Michaelis-Menten constant
- hCE
- human carboxylesterase
- kcat
- turnover number
- Ki
- competitive inhibition constant
- DEA
- N,N-diisopropylethylamine
- DTT
- dithiothreitol
- SDS-PAGE
- sodium dodecyl sulfate-polyacrylamide gel electrophoresis
- Received March 7, 1997.
- Accepted June 5, 1997.
- The American Society for Pharmacology and Experimental Therapeutics
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